Project Abstract We have found that a protein made exclusively in the antrum of the stomach, called gastrokine-1 (Gkn1), is an essential regulator of diet-induced obesity. Our overall goals are to block Gkn1 to reduce or prevent obesity, and to determine the mechanism of Gkn1 regulation of obesity. Gkn1 expression is constitutive and abundant (1% of total stomach RNA) and the resulting 18kDa protein is highly resistant to denaturation by proteases, detergents and chaotropic agents. Gkn1 is secreted along with mucus into the stomach lumen and travels the entire length of the gastrointestinal tract intact. Gkn1 decorates microbes in the lumen of the colon, suggesting that Gkn1 effects on obesity might be regulated by its interaction with gut microbes. Indeed Gkn1-/- mice, on a high fat diet (HFD), are resistant to weight gain and resistant to changes in the intestinal microbiome that typically occur on a HFD. Gkn1+/- (control) mice exhibit increased percentages of Firmicutes and Proteobacteria, and reduction of Bacteroidetes, on a HFD and this does not occur in Gkn1-/- mice. We also find that adult Gkn1-/- mice have significant quantities of brown/beige fat in all of their fat pads, which is not observed in Gkn1+/- mice. Thus Gkn1, made in the stomach, binds to microbes in the distal gut and modulates diet-induced alterations in the microbiome, fat metabolism, and obesity. Since Gkn1 is a protein found in the lumen of the gut, our first hypothesis is that blocking Gkn1 will stabilize the gut microbiome and prevent diet- induced obesity. Gkn1 protein prevents assembly of amyloid fibers, which are a structural feature found in many proteins. Amyloids are produced by many bacteria to facilitate biofilm formation and adherence. Since we observe Gkn1 decorating the outside of microbes, our second hypothesis is that Gkn1binds to bacterial amyloids to prevent adherence and/or biofilm formation. To test these hypotheses we will address the following aims: 1. Identify therapies that block Gkn1 regulation of the microbiome and diet-induced obesity in vivo. 2. Examine the effect of Gkn1 on microbial biofilm formation and adherence. These studies will delineate how a food supplement (egg meal) can be effective for treatment of obesity and will identify mechanisms whereby Gkn1 regulates intestinal microbes, which will provide direction for future efforts to manipulate the microbiome to improve health. In addition, the concept that the stomach plays a key role on the regulation of the distal gut microbiome and obesity is entirely unprecedented and will have a direct impact on current surgical and mediacl therapies for obesity.